Fusion protein comprising diphtheria toxin non-toxic mutant CRM197 or fragment thereof

ABSTRACT

Provided in the present invention are a diphtheria toxin non-toxic mutant CRM197 or a fragment thereof as an adjuvant in a fusion protein and the use thereof to enhance the immunogenicity of a target protein fused therewith, for example, an HEV capsid protein, or an influenza virus M2 protein or an immunogenic fragment thereof. Also provided is a method for enhancing the immunogenicity of a target protein, comprising the fusion expression of the CRM197 or the fragment thereof with the target protein to form a fusion protein. Further provided is a fusion protein comprising the CRM197 or the fragment thereof and a target protein, the CRM197 or the fragment thereof enhancing the immunogenicity of the target protein. The present invention also provides an isolated nucleic acid encoding the fusion protein, a construct and a vector comprising said nucleic acid, and a host cell comprising the nucleic acid.

This application incorporates by reference the contents of a 102 kb textfile created on Sep. 27, 2016 and named“00768500044substitutesequencelisting.txt,” which is the sequencelisting for this application.

FIELD OF THE INVENTION

The invention relates to the field of molecular virology and immunology.In particular, the invention relates to a use of a diphtheria toxinnon-toxic mutant CRM197 or a fragment thereof as intramolecular adjuvantin a fusion protein for enhancing immunogenicity of a target proteinfused thereto (for example, an HEV capsid protein, an influenza virus M2protein or an immunogenic fragment thereof). The invention also relatesto a method for enhancing immunogenicity of a target protein (forexample, an HEV capsid protein, an influenza virus M2 protein or animmunogenic fragment thereof), comprising the fusion expression ofCRM197 or a fragment thereof with the target protein to form a fusionprotein. The invention also relates to a fusion protein comprisingCRM197 or a fragment thereof and a target protein (for example, an HEVcapsid protein, an influenza virus M2 protein or an immunogenic fragmentthereof), wherein said CRM197 or a fragment thereof enhancesimmunogenicity of the target protein. The invention also relates to anisolated nucleic acid encoding the fusion protein, a construct and avector comprising the nucleic acid, and a host cell comprising thenucleic acid. The invention also relates to a use of the fusion proteinin the manufacture of a pharmaceutical composition or a vaccine.

BACKGROUND OF THE INVENTION

Diphtheria toxin (DT) has been deeply studied. The studies on structureshow that diphtheria toxin consists of three domains: N-terminalCatalytic Domain C (aa 1-190, C domain) (also called Fragment A), middleTransmembrane Domain T (aa 201-384, T domain), and C-terminal ReceptorBinding domain R (aa 386-535, R domain) (Choe S, Bennett M, Fujii G, etal., Nature. 1992. 357:216-222). ONTAK (DAB389-IL-2), prepared by fusionof the former two domains of diphtheria toxin with interleukin 2 (IL-2),was approved by FDA on the market in 1999, for the treatment of adultcutaneous T-cell lymphoma. This demonstrates that the three domains ofdiphtheria toxin may be used separately and play their own roles,respectively.

CRM197 (Cross-Reacting Materials 197) is a diphtheria toxin non-toxicmutant (Uchida, T., A. M, Pappenheimer, Jr., R. Gregory, et al., J.Biol. Chem. 1973. 248:3838-3844), which differs from a wild-type geneencoding DT by a single nucleotide mutation, resulting in the amino acidresidue at position 52 changed from Gly to Glu (G. Giannini, R.Rappuoli, G. Ratti et al., Nucleic Acids Research. 1984. 12: 4063-4070).

Studies show that although CRM197 has a structure similar to that of awild-type DT (namely, having said three domains), its Fragment A losesthe ability of binding to NAD, is unable to bind to EF2 and therebyloses the cytoxicity possessed by natural DT, indicating that the aminoacid residue Gly at position 52 plays an important role in the bindingof DT to NAD (K. Moyner, G. Christiansen, Acta path microbialimmunolscand sect C. 1984, 92:17-23). Although CRM197 loses thecytotoxicity, it retains a stronge immunogenicity comparable to that ofa wild-type DT. Therefore, CRM197 is generally used as a protein carrierfor crosslinking other haptens so as to prepare conjugate vaccines.

As early as 1985, Porter et al., crosslinked polysaccharides on Hibsurface to CRM197 and DT protein carrier, respectively, and preparedthem into vaccines, and studied the difference of them inimmunogenicity. The experimental results showed that there was nosignificant difference between the two crosslinked vaccines in terms ofimmune effect, both of them could stimulate the generation of a strongimmune response and immunologic memory in infants (Porter Anderson,Micheal E. Pichichero and Richard A. J. Clin. Invest. 1985: 52-59).After comparing pneumococcal conjugate vaccines crosslinked to variousproteins, it is found that the vaccines wherein CRM197 is used as aprotein carrier have a good immune effect in animal experiments andclinical trials, and CRM197 is safe without a side-effect of toxicity(Black, S., H. Shinefield, et al. Pediatr Infect Dis J, 2000, 19(3);187-195). In current, pneumococcal conjugate vaccines, in which CRM197is used as a protein carrier, mainly refer to PCV7, PCV9, PCV13, and thelike. The results of clinical trials showed that these vaccines had goodimmunogenicity and safety in children less than two years old(Barricarte, A., J. Castilla, et al. Clin Infect Dis, 2007, 44(11):1436-1441; Madhi, S., P. Adrian, et al. Vaccine, 2007, 25(13):2451-2457; Duggan, S. T. Drugs, 2010, 70(15): 1973-1986). Epidemicmeningitis conjugate vaccines can be prepared by crosslinking CRM197 topolysaccharides on surface of N. menigitidis. For example, vaccines suchas Meningitec (Wyeth Pharmaceuticals), Menjugate (Novartis vaccines),and Menveo (Novartis vaccines), in which CRM197 is used as a proteincarrier, have been commercially available.

Although CRM197 loses enzymatic activity and cytotoxity, it is stillable to bind to a specific receptor of DT, i.e. heparin-binding EGF-likegrowth factor (HB-EGF). Since the expression of the receptor isgenerally up-regulated in cancerous tissues, like DT, CRM197 also hasanti-tumor effect (Buzzi, S., D. Rubboli, et al. Immunotherapy, 2004,53(11)). The studies also found that CRM197 could pass throughBlood-Brain-Barrier (BBB), and therefore could be used as a carrier fordelivery of drugs to brain (Gaillard, P. J., and A. G. de Boer. JControl Release, 2006, 116(2): 60-62).

Although it has been reported that CRM197 has multiple functions, inparticular, has a strong immunogenicity and can be used asimmunoadjuvant, it is not reported yet that CRM197 may be used asintramolecular adjuvant for enhancing immunogenicity of a target proteinfused thereto in a fusion protein. The invention uses Hepatitis E capsidprotein as an example, and demonstrates for the first time that CRM197or a fragment thereof can enhance immunogenicity of a protein fusedthereto in a fusion protein, and thereby can be used as intramolecularadjuvant.

DESCRIPTION OF THE INVENTION

In the invention, unless otherwise specified, the scientific andtechnical terms used herein have the meanings as generally understood bya person skilled in the art. Moreover, the laboratory operations of cellculture, molecular genetics, nucleic acid chemistry, biologicalchemistry, and immunology used herein are the routine operations widelyused in the corresponding fields. Meanwhile, for the purpose of betterunderstanding of the invention, the definitions and explanations of therelevant terms are provided as follows.

According to the invention, the term “CRM197” refers to a diphtheriatoxin non-toxic mutant, which differs from a wild-type diphtheria toxinby an amino acid residue at position 52 changed from Gly to Glu (G.Giannini, R. Rappuoli, G. Ratti et al., Nucleic Acids Research. 1984.12: 4063-4070). Diphtheria toxin is well known by a person skilled inthe art (see, for example, Choe S, Bennett M, Fujii G, et al., Nature.1992. 357:216-222), whose amino acid sequence may be found by referenceto GenBank accession No. AAV70486.1.

In the invention, the exemplary amino acid sequence of CRM197 is setforth in SEQ ID NO: 2. Therefore, in the invention, when the sequence ofCRM197 is involved, it is described as the sequence set forth in SEQ IDNO:2. For example, in the expression “amino acid residues from positions1 to 190 of CRM197”, amino acid residues from positions 1 to 190 refersto amino acid residues from positions 1 to 190 of SEQ ID NO: 2. However,a person skilled in the art understands that mutations or variations(including, but not limited to, substitution, deletion and/or addition)may naturally occur in or are introduced artificially into SEQ ID NO: 2without affecting the biological properties of CRM197. Therefore, in theinvention, the term “CRM197” intends to comprise all such polypeptidesand variants, including the polypeptide set forth in SEQ ID NO: 2 andits natural or artificial variants, wherein the variants retain thebiological properties of CRM197, i.e. have a strong immunogenicity andno cytotoxity. In addition, when sequence fragments of CRM197 aredescribed, they include not only the sequence fragments of a polypeptideset forth in SEQ ID NO: 2, but also the corresponding sequence fragmentsof the natural or artificial variants of the polypeptide. For example,the expression “amino acid residues from positions 1 to 190 of CRM197”intends to comprise amino acid residues from positions 1 to 190 of SEQID NO: 2 and the corresponding fragments of the variants (natural orartificial) of a polypeptide set forth in SEQ ID NO: 2.

According to the invention, an Hepatitis E virus (HEV) capsid proteinrefers to a protein encoded by HEV ORF2. The sequence of HEV ORF2 iswell known in the art (see, for example, DDBJ accession No. D11092). Inthe invention, when the sequence of HEV ORF2 is involved, it isdescribed as the sequence set forth in DDBJ accession No. D11092. Forexample, in the expression “amino acid residues from positions 368 to606 of a polypeptide encoded by HEV ORF2”, amino acid residues frompositions 368 to 606 refers to amino acid residues from positions 368 to606 of a polypeptide encoded by D11092. However, a person skilled in theart understands that mutations or variations (including, but not limitedto, substitution, deletion and/or addition) may naturally occur in orare introduced artificially into HEV ORF2 or a polypeptide encodedthereby without affecting the biological properties thereof (such asantigenicity and immunogenicity). Therefore, in the invention, the term“HEV ORF2” intends to comprise all such polypeptides and variants,including the sequence set forth in D11092 and its natural or artificialvariants. In addition, when sequence fragments of HEV ORF2 (or apolypeptide encoded thereby) are described, they include not only thesequence fragments of D11092 (or a polypeptide encoded thereby), butalso the corresponding sequence fragments of the natural or artificialvariants of D11092 (or a polypeptide encoded thereby). For example, theexpression “amino acid residues from positions 368 to 606 of apolypeptide encoded by HEV ORF2” intends to comprise amino acid residuesfrom positions 368 to 606 of a polypeptide encoded by D11092 and thecorresponding fragments of the variants (natural or artificial) of apolypeptide encoded by D11092. The exemplary amino acid sequence of anHEV capsid protein (a polypeptide encoded by ORF2 of D11092) isdescribed in SEQ ID NO: 31.

According to the invention, an influenza virus M2 protein refers to aprotein encoded by the seventh segment of type A or type B influenzavirus genome or a protein encoded by the sixth segment of type Cinfluenza virus genome. The exemplary amino acid sequence of aninfluenza virus M2 protein is described in SEQ ID NO: 32.

According to the invention, the expression “corresponding sequencefragments” or “corresponding fragments” refers to fragments that arelocated in equal positions of sequences when the sequences are subjectedto optimal alignment, namely, the sequences are aligned to obtain ahighest percentage of identity.

According to the invention, when used in the background ofproteins/polypeptides, the term “variant” refers to a protein, whoseamino acid sequence is different from a reference protein/polypeptide(for example, CRM197 of the invention) by one or more (for example,1-10, or 1-5 or 1-3) amino acids (such as conservative amino acidsubstitutions), or which has an identity of at least 60%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% to a reference protein/polypeptide (forexample, CRM197 of the invention), and which retains the essentialcharacteristics of the reference protein/polypeptide. In the invention,the essential characteristics of CRM197 may refer to a strongimmunogenicity and no cytotoxity, and the essential characteristics ofan HEV capsid protein and an influenza virus M2 protein may refer toantigenicity and/or immunogenicity thereof.

According to the invention, the term “identity” refers to the matchdegree between two polypeptides or between two nucleic acids. When twosequences for comparison have the same base or amino acid monomersub-unit at a certain site (e.g., each of two DNA molecules has anadenine at a certain site, or each of two polypeptides has a lysine at acertain site), the two molecules are identical at the site. The percentidentity between two sequences is a function of the number of identicalsites shared by the two sequences over the total number of sites forcomparison×100. For example, if 6 of 10 sites of two sequences arematched, these two sequences have an identity of 60%. For example, DNAsequences: CTGACT and CAGGTT share an identity of 50% (3 of 6 sites arematched). Generally, the comparison of two sequences is conducted in amanner to produce maximum identity. Such alignment can be conducted byusing a computer program such as Align program (DNAstar, Inc.) which isbased on the method of Needleman, et al. (J. Mol. Biol. 48:443-453,1970). The percent identity between two amino acid sequences can bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the algorithmof Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

As used in the invention, the term “conservative substitution” refers toamino acid substitutions which would not negatively affect or change theessential characteristics of a protein/polypeptide comprising the aminoacid sequence. For example, a conservative substitution may beintroduced by standard techniques known in the art such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions include substitutions wherein an amino acid residue issubstituted with another amino acid residue having a similar side chain,for example, with a residue similar to the corresponding amino acidresidue physically or functionally (such as, having similar size, shape,charges, chemical properties including the capability of formingcovalent bond or hydrogen bond, etc.). The families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids having alkaline side chains (for example,lysine, arginine and histidine), amino acids having acidic side chains(for example, aspartic acid and glutamic acid), amino acids havinguncharged polar side chains (for example, glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), aminoacids having nonpolar side chains (for example, alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), amino acidshaving β-branched side chains (such as threonine, valine, isoleucine)and amino acids having aromatic side chains (for example, tyrosine,phenylalanine, tryptophan, histidine). Therefore, an amino acid residueis preferably substituted with another amino acid residue from the sameside-chain family. Methods for identifying amino acid conservativesubstitutions are well known in the art (see, for example, Brummell etal., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng.12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

According to the invention, the term “immunogenicity” refers to anability of stimulating the formation of specific antibodies orsensitized lymphocytes in organisms. It not only refers to the propertyof an antigen to stimulate a specific immunocyte to activate,proliferate and differentiate the immunocyte so as to finally generateimmunologic effector substance such as antibodies and sensitizedlymphocytes, but also refers to the specific immune response whereinantibodies or sensitized T lymphocytes can be formed in immune system ofan organism after stimulating the organism with an antigen.Immunogenicity is the most important property of an antigen. Whether anantigen can successfully induce the generation of an immune response ina host depends on three factors, properties of an antigen, reactivity ofa host, and immunization means.

According to the invention, the term “immunogenic fragment” refers tosuch a polypeptide fragment, which at least partially retains theimmunogenicity of the protein from which it is derived. For example,immunogenic fragments of an HEV capsid protein refer to fragments of anHEV capsid protein which at least partially retain immunogenicity, forexample, HEV-239, E2 or E2s as described in the invention (see, Li etal., J Biol Chem. 280(5): 3400-3406 (2005); Li et al., PLoS Pathogens.5(8): e1000537 (2009)); immunogenic fragments of an influenza virus M2protein refer to fragments of M2 protein which at least partially retainimmunogenicity, for example, M2e as described in the invention (see,Fiers W et al., Vaccine. 27(45):6280-6283(2009)).

According to the invention, HEV-239 (or 239 in brief) refers to apolypeptide consisting of amino acid residues from positions 368 to 606of a polypeptide encoded by HEV ORF2 (i.e. HEV capsid protein); E2refers to a polypeptide consisting of amino acid residues from positions394 to 606 of a polypeptide encoded by HEV ORF2; E2s refers to apolypeptide consisting of amino acid residues from positions 455 to 606of a polypeptide encoded by HEV ORF2.

According to the invention, the term “M2e” refers to a polypeptideconsisting of amino acid residues from positions 1 to 24 of an influenzavirus M2 protein.

In the invention, the term “polypeptide” and “protein” have the samemeanings and may be used interchangeably. Moreover, in the invention,amino acids are generally represented by one letter code andthree-letter code well known in the art. For example, alanine may berepresented by A or Ala.

According to the invention, the term “E. coli expression system” refersto an expression system consisting of E. coli (strain) and a vector,wherein the E. coli (strain) is available on the market, including butnot limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3), etc.

According to the invention, the term “vector” refers to a nucleic acidvehicle which can have a polynucleotide inserted therein. When thevector allows for the expression of the protein encoded by thepolynucleotide inserted therein, the vector is called an expressionvector. The vector can be introduced into the host cell bytransformation, transduction, or transfection, and have the carriedgenetic material elements expressed in a host cell. Vectors are wellknown by a person skilled in the art, including, but not limited toplasmids, phages, cosmids and the like.

According to the invention, the term “chromatography” includes, but isnot limited to: ion exchange chromatography (e.g. cation-exchangechromatography), hydrophobic interaction chromatography, absorbentchromatography (e.g. hydroxyapatite chromatography), gel filtrationchromatography (gel exclusion chromatography), and affinitychromatography.

According to the invention, the term “pharmaceutically acceptablecarriers and/or excipients” refers to carriers and/or excipients thatare pharmacologically and/or physiologically compatible with subjectsand active ingredients, and are well known in the art (see, for example,Remington's Pharmaceutical Sciences. Edited by Gennaro A R, 19th ed.Pennsylvania: Mack Publishing Company, 1995), including, but not limitedto pH adjusting agents, surfactants, adjuvants, and ionic strengthenhancers. For example, pH adjusting agents include, but are not limitedto, phosphate buffers; surfactants include, but are not limited to:anion surfactants, cation surfactants, or non-ionic surfactants (forexample, Tween-80); and ionic strength enhancers include, but are notlimited to sodium chloride.

According to the invention, the term “adjuvant” refers to a non-specificimmuno-potentiator, which can enhance immune response to an antigen orchange the type of immune response in an organism when it is deliveredtogether with the antigen to the organism or is delivered to theorganism in advance. There are a variety of adjuvants, including butlimited to, aluminium adjuvants (for example, aluminum hydroxide),Freund's adjuvants (for example, Freund's complete adjuvant and Freund'sincomplete adjuvant), corynebacterium parvum, lipopolysaccharide,cytokines, and the like. Freund's adjuvant is the most commonly usedadjuvant in animal experiments currently. Aluminum hydroxide adjuvant ismore commonly used in clinical trials.

According to the invention, the term “intramolecular adjuvant” refers tosuch an adjuvant, which forms a fusion protein with a target protein(i.e. an antigen), is present in the same molecule as the antigen (i.e.a fusion protein comprising it and the antigen), and acts as theadjuvant of the antigen to enhance immunogenicity of the antigen.Namely, an intramolecular adjuvant is an adjuvant capable of enhancingimmunogenicity of a target protein (antigen) fused and expressedtherewith, which generally refers to a polypeptide fragment. In theinvention, an intramolecular adjuvant especially refers to a diphtheriatoxin non-toxic mutant CRM197 or a fragment thereof.

The techniques for forming a fusion protein by fusion expression of twoor more proteins are well known in the art (see, for example, Sambrook Jet al., Molecular Cloning: A Laboratory Manual (Second Edition), ColdSpring Harbor Laboratory Press, 1989; and F. M. Ausubel et al., ShortProtocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc.,1995). Generally, DNA fragments encoding two or more proteins are linkedtogether in frame by recombinant DNA techniques, and a fusion protein isobtained by protein expression. Optionally, a linker may be used or notin fusion expression of two or more proteins.

According to the invention, the term “linker” refers to a short peptidefor linking two molecules (for example, proteins). Generally, a fusionprotein, such as a target protein 1-linker-a target protein 2, isobtained by introduction (for example, by PCR amplification or ligase)of a polynucleotide encoding the short peptide between two DNA fragmentsencoding two target proteins to be linked, respectively, and proteinexpression thereof. As well known by a person skilled in the art,linkers include, but are not limited to flexible linking peptides, suchas Gly-Gly-Gly-Gly (SEQ ID NO:57), Gly-Gly-Gly-Gly-Ser (SEQ ID NO:58),Gly-Gly-Ser-Ser (SEQ ID NO:59) and (Gly-Gly-Gly-Gly-Ser)₃ (SEQ IDNO:60).

According to the invention, the term “an effective amount” refers to anamount that is sufficient to achieve or at least partially achieve theexpected effect. For example, an amount effective for preventing adisease (such as HEV or influenza virus infection) refers to an amounteffective for preventing, suppressing, or delaying the occurrence of adisease (such as HEV or influenza virus infection). An effective amountfor treating a disease refers to an amount effective for curing or atleast partially blocking a disease and its complication in a patientwith the disease. The determination of such an effective amount iswithin the ability of a person skilled in the art. For example, anamount effective for a therapeutic use depends on severity of a diseaseto be treated, general state of the immune system in a patient, generalconditions of a patient, such as age, weight and gender, administrationmeans of drugs, additional therapies used simultaneously, and the like.

The invention is at least partially based on the inventors' surprisingdiscovery: after fusion expression of CRM197 or a fragment thereof witha target protein (for example, an HEV capsid protein, an influenza virusM2 protein or an immunogenic fragment thereof), CRM197 or a fragmentthereof significantly enhances immunogenicity of the target protein.Namely, CRM197 or a fragment thereof may be used as intramolecularadjuvant for enhancing immunogenicity of a target protein by fusionexpression with the target protein.

Therefore, in one aspect, the invention relates to a fusion proteincomprising CRM197 or a fragment thereof and a target protein, whereinsaid CRM197 or a fragment thereof enhances immunogenicity of the targetprotein.

In a preferred embodiment, the fragment of CRM197 comprises, forexample, Catalytic Domain C (aa 1-190, also called Fragment A in thepresent application), Transmembrane Domain T (aa 201-384), and/orReceptor Binding domain R (aa 386-535) of CRM197. For example, thefragment of CRM197 may comprise Fragment A, or Fragment A andTransmembrane Domain T.

In another preferred embodiment, the fragment of CRM197 comprises aa1-190 of CRM197, for example, comprises aa 1-389 of CRM197. In anotherpreferred embodiment, the fragment of CRM197 consists of aa 1-190 or aa1-389 of CRM197. In the present application, the exemplary amino acidsequence of CRM197 is set forth in SEQ ID NO: 2, and the correspondingnucleotide sequence is set forth in SEQ ID NO:1.

In a preferred embodiment, the target protein may be an HEV capsidprotein, an influenza virus M2 protein, or an immunogenic fragmentthereof. In another preferred embodiment, the immunogenic fragment of anHEV capsid protein may comprise or be, for example, HEV-239 (aa 368-606of the HEV capsid protein), E2 (aa 394-606 of the HEV capsid protein) orE2s (aa 455-606 of the HEV capsid protein), and the like. In anotherpreferred embodiment, the immunogenic fragment of a M2 protein maycomprise or be, for example, M2e (aa 1-24 of the M2 protein).

In a preferred embodiment, in the fusion protein of the invention,CRM197 or a fragment thereof may be linked to the N-terminus and/orC-terminus of the target protein, optionally via a linker. The linkerfor linking two peptide fragments are well known in the art, includingbut not limited to flexible linking peptides, such as Gly-Gly-Gly-Gly(SEQ ID NO:57), Gly-Gly-Gly-Gly-Ser (SEQ ID NO:58), Gly-Gly-Ser-Ser (SEQID NO:59) and (Gly-Gly-Gly-Gly-Ser)₃ (SEQ ID NO:60), etc. Such linkersare well known in the art, and the selection thereof is within theability of a person skilled in the art.

In a preferred embodiment, the fusion protein of the invention maycomprise CRM197 or a fragment thereof, and a HEV capsid protein or animmunogenic fragment thereof, which are linked together, optionally viaa linker. For example, the fusion protein of the invention may be aprotein having an amino acid sequence set forth in SEQ ID NO: 4, 6, 8,10, 12, 14, 16 or 18.

In a preferred embodiment, the fusion protein of the invention maycomprise CRM197 or a fragment thereof, and an influenza virus M2 proteinor an immunogenic fragment thereof, which are linked together,optionally via a linker. For example, the fusion protein of theinvention may be a protein having an amino acid sequence set forth inSEQ ID NO:34, 36, 38, 40, 42 or 44.

In the fusion protein of the invention, CRM197 or a fragment thereofsuperisingly enhances immunogenicity of a target protein (such as HEVcapsid protein, an influenza virus M2 protein or or an immunogenicfragment thereof) fused thereto (optionally via a linker) significantly,and thus may be used as intramolecular adjuvant.

In another aspect, the invention provides a polynucleotide encoding thefusion protein as defined above, and also provides a constructcomprising the polynucleotide.

In another aspect, the invention provides a vector comprising: apolynucleotide encoding the fusion protein as defined above or aconstruct comprising the polynucleotide. The vector of the invention maybe a cloning vector, or an expression vector.

In a preferred embodiment, the vector of the invention may be, forexample, plasmid, cosmid, phage, and the like.

In another aspect, the invention provides a host cell or organismcomprising the polynucleotide, the construct, or the vector of theinvention. Said host cell includes, but is not limited to, prokaryoticcell such as E. coli cell, and eukaryotic cell such as yeast cell,insect cell, plant cell and animal cell (such as mammalian cell, forexample mice cell, human cell and the like). The cell of the inventionmay be a cell line, such as 293T cell. In an embodiment, the organism isplant or animal.

In another aspect, the invention also relates to a pharmaceuticalcomposition or vaccine comprising the fusion protein of the invention,and optionally a pharmaceutically acceptable carrier and/or excipient.Depending on the target protein used in the fusion protein, thepharmaceutical composition or vaccine of the invention may be useful forthe prevention and/or treatment of various diseases (i.e. diseases thatcan be prevented or treated by the target protein). For example, whenthe target protein used is an HEV capsid protein or an immunogenicfragment thereof, the pharmaceutical composition of the invention may beused to prevent and/or treat HEV infection and diseases associated withHEV infection such as Hepatitis E; when the target protein used is aninfluenza virus M2 protein or an immunogenic fragment thereof, thepharmaceutical composition of the invention may be used to preventand/or treat influenza virus infection and diseases associated withinfluenza virus infection such as influenza.

In another aspect, the invention also relates to a use of the fusionprotein of the invention in the manufacture of a pharmaceuticalcomposition for the prevention and/or treatment of diseases that can beprevented or treated by the target protein. Depending on the targetprotein used in the fusion protein, the pharmaceutical composition ofthe invention may be used to prevent and/or treat various diseases. Forexample, when the target protein used is an HEV capsid protein or animmunogenic fragment thereof, the pharmaceutical composition of theinvention may be used to prevent and/or treat HEV infection and diseasesassociated with HEV infection such as Hepatitis E; when the targetprotein used is an influenza virus M2 protein or an immunogenic fragmentthereof, the pharmaceutical composition of the invention may be used toprevent and/or treat influenza virus infection and diseases associatedwith influenza virus infection such as influenza.

In another aspect, the invention also relates to a method for preventingand/or treating HEV infection and/or diseases associated with HEVinfection such as Hepatitis E, comprising administering an effectiveamount of the fusion protein of the invention or the pharmaceuticalcomposition comprising the fusion protein, wherein the fusion proteincomprises CRM197 or a fragment thereof and an HEV capsid protein or animmunogenic fragment thereof, which are linked together, optionally viaa linker.

In another aspect, the invention also relates to a method for preventingand/or treating influenza virus infection and diseases associated withinfluenza virus infection such as influenza, comprising administering aneffective amount of the fusion protein of the invention or thepharmaceutical composition comprising the fusion protein, wherein thefusion protein comprises CRM197 or a fragment thereof and an influenzavirus M2 protein or an immunogenic fragment thereof, which are linkedtogether, optionally via a linker.

In another aspect, the invention provides a method for enhancingimmunogenicity of a target protein, comprising obtaining a fusionprotein comprising CRM197 or a fragment thereof as defined above and thetarget protein, so as to enhance immunogenicity of the target protein.

In a preferred embodiment, the fusion protein may be obtained by fusionexpression of CRM197 or a fragment thereof with the target protein,optionally using a linker. In a preferred embodiment, the target proteinis the HEV capsid protein, the influenza virus M2 protein or animmunogenic fragment thereof as described above.

Therefore, in an embodiment, the invention provides a method forenhancing immunogenicity of an HEV capsid protein or an immunogenicfragment thereof, comprising obtaining a fusion protein comprisingCRM197 or a fragment thereof and an HEV capsid protein or an immunogenicfragment thereof, so as to enhance immunogenicity of the HEV capsidprotein or an immunogenic fragment thereof. In a preferred embodiment,the fusion protein may be obtained by fusion expression of CRM197 or afragment thereof with an HEV capsid protein or an immunogenic fragmentthereof, optionally using a linker.

In another embodiment, the invention provides a method for enhancingimmunogenicity of an influenza virus M2 protein or an immunogenicfragment thereof, comprising obtaining a fusion protein comprisingCRM197 or a fragment thereof and an influenza virus M2 protein or animmunogenic fragment thereof, so as to enhance immunogenicity of theinfluenza virus M2 protein or an immunogenic fragment thereof. In apreferred embodiment, the fusion protein may be obtained by fusionexpression of CRM197 or a fragment thereof with an influenza virus M2protein or an immunogenic fragment thereof, optionally using a linker.

In another aspect, the invention relates to a use of CRM197 or afragment thereof in the enhancement of immunogenicity of a targetprotein, characterized by obtaining a fusion protein comprising CRM197or a fragment thereof and the target protein.

In a preferred embodiment, the fusion protein may be obtained by fusionexpression of CRM197 or a fragment thereof with the target protein,optionally using a linker. In a preferred embodiment, the target proteinis an HEV capsid protein, an influenza virus M2 protein, or animmunogenic fragment thereof.

Therefore, in an embodiment, the invention relates to a use of CRM197 ora fragment thereof in the enhancement of immunogenicity of an HEV capsidprotein or an immunogenic fragment thereof, characterized by obtaining afusion protein comprising CRM197 or a fragment thereof and the HEVcapsid protein or an immunogenic fragment thereof. In a preferredembodiment, the fusion protein may be obtained by fusion expression ofCRM197 or a fragment thereof with the HEV capsid protein or animmunogenic fragment thereof, optionally using a linker.

In another embodiment, the invention relates to a use of CRM197 or afragment thereof in the enhancement of immunogenicity of an influenzavirus M2 protein or an immunogenic fragment thereof, characterized byobtaining a fusion protein comprising CRM197 or a fragment thereof andthe influenza virus M2 protein or an immunogenic fragment thereof. In apreferred embodiment, the fusion protein may be obtained by fusionexpression of CRM197 or a fragment thereof with the influenza virus M2protein or an immunogenic fragment thereof, optionally using a linker.

Beneficial Effect of the Invention

The invention demonstrates for the first time that CRM197 and fragmentsthereof may be used as intramolecular adjuvant for enhancingimmunogenicity of a target protein. Therefore, the invention provides anovel use of CRM197 and fragments thereof, and provides a novel methodfor enhancing immunogenicity of a target protein.

In addition, since the fusion protein of the invention exhibits astronger immunogenicity as compared to a target protein alone, theinvention provides a new option for the manufacture of a medicament orvaccine and may achieve more effective treatment and prevention of thecorresponding diseases.

For example, the fusion protein of the invention comprising CRM197 (or afragment thereof) and an HEV capsid protein (or an immunogenic fragmentthereof) exhibits a stronger immunogenicity as compared to a HEV capsidprotein (or an immunogenic fragment thereof) alone, and therefore thefusion protein may be useful for the manufacture of a pharmaceuticalcomposition and more effectively prevent and treat HEV infection anddiseases associated with HEV infection such as Hepatitis E.

For example, the fusion protein of the invention comprising CRM197 (or afragment thereof) and an influenza virus M2 protein (or an immunogenicfragment thereof) exhibits a stronger immunogenicity as compared to ainfluenza virus M2 protein (or an immunogenic fragment thereof) alone,and therefore the fusion protein may be useful for the manufacture of apharmaceutical composition and more effectively prevent and treatinfluenza virus infection and diseases associated with influenza virusinfection such as influenza. For example, when M2e protein is fused tothe N-terminal of CRM197 (or a fragment thereof), the fusion proteinthus formed may form a tetramer or other polymer configuration, and hasa good reactivity with a protective monoclonal antibody 019 (see, Fu etal., Virology, 2009, 385:218-226) in vitro (see, FIG. 12B), and has agood immunogenicity in vivo (see, FIG. 14). Therefore, the fusionprotein thus formed is useful for developing general influenza vaccines.

Description of Sequence Information

The information of the sequences as involved in the invention isprovided in the following table.

SEQ SEQ ID ID NO: Depiction NO: Depiction 1 the nucleotide sequence of 2the amino acid sequence CRM197 of CRM197 3 the nucleotide sequence of 4the amino acid sequence CRM197-L-E2 of CRM197-L-E2 5 the nucleotidesequence of 6 the amino acid sequence CRM197-L-E2s of CRM197-L-E2s 7 thenucleotide sequence of 8 the amino acid sequence 389-L-E2 of 389-L-E2 9the nucleotide sequence of 10 the amino acid sequence 389-L-E2s of389-L-E2s 11 the nucleotide sequence of 12 the amino acid sequenceA-L-E2 of A-L-E2 13 the nucleotide sequence of 14 the amino acidsequence A-L-E2s of A-L-E2s 15 the nucleotide sequence of 16 the aminoacid sequence 389-E2s of 389-E2s 17 the nucleotide sequence of 18 theamino acid sequence A-E2s of A-E2s 19 primer CRM197F 20 primer CRM197R21 primer CRM197-linker R 22 primer 389-linker R 23 primer A-linker R 24primer E2F 25 primer E2sF 26 primer Drp59R 27 primer 389-E2s R 28 primerA-E2s R 29 primer 389-E2s F 30 primer A-E2s F 31 the amino acid sequenceof 32 the amino acid sequence of HEV capsid protein M2 protein 33 thenucleotide sequence of 34 the amino acid sequence of CRM197-L-M2eCRM197-L-M2e 35 the nucleotide sequence of 36 the amino acid sequence of389-L-M2e 389-L-M2e 37 the nucleotide sequence of 38 the amino acidsequence of A-L-M2e A-L-M2e 39 the nucleotide sequence of 40 the aminoacid sequence of M2e-L-CRM197 M2e-L-CRM197 41 the nucleotide sequence of42 the amino acid sequence of M2e-L-389 M2e-L-389 43 the nucleotidesequence of 44 the amino acid sequence of M2e-L-A M2e-L-A 45 primerCRM197F1 46 primer CRM197-linker R1 47 primer 389-linker R1 48 primerA-linker R1 49 primer M2eF1 50 primer M2eR 51 primer M2eF2 52 primerM2e-Linker R 53 primer CRM197F2 54 primer CRM197 R2 55 primer 389 R 56primer A R

The embodiments of the invention are further described in detail byreference to the drawings and examples. However, a person skilled in theart would understand that the following drawings and examples areintended for illustrating the invention only, rather than defining thescope of the invention. According to the detailed depiction of thefollowing drawings and preferred embodiments, various purposes andadvantages of the invention would be obvious for a person skilled in theart.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the clone design of the fusion proteins constructed inExample 2, wherein the linker used (Linker, also referred to L for shortin the present application) is a flexible fragment consisting of 15amino acid residues, whose sequence is GGGGSGGGGSGGGGS (SEQ ID NO:60);the CRM197 used comprised 535 amino acids, whose sequence is set forthin SEQ ID NO: 2; 389 refers to a polypeptide comprising amino acidresidues from positions 1 to 389 (aa 1-389) of CRM197; A refers to apolypeptide comprising amino acid residues from positions 1 to 190 (aa1-190) of CRM197; E2 refers to a polypeptide comprising amino acidresidues from positions 394 to 606 (aa 394-606) of an HEV capsidprotein; E2s refers to a polypeptide comprising amino acid residues frompositions 455 to 606 (aa 455-606) of an HEV capsid protein.

FIGS. 2A-2D show SDS-PAGE analytic results of expression, purificationand renaturation of the fusion proteins constructed in Example 2,wherein the sample used in FIG. 2A is the precipitate (i.e. inclusionbody) obtained by centrifuging the disrupted bacteria afterultrasonication, the sample used in FIG. 2B is a 4M urea dissolvedsupernatant, the sample used in FIG. 2C is a 8M urea dissolvedsupernatant, and the sample used in FIG. 2D is a protein renatured intoPBS. Lane M: protein molecular weight marker; Lane 1: CRM197-L-E2; Lane2: CRM197-L-E2s; Lane 3: 389-L-E2; Lane 4: 389-L-E2s; Lane 5: 389-E2s;Lane 6: A-L-E2; Lane 7: A-L-E2s; Lane 8: A-E2s. The results showed thatall the constructed fusion proteins could be expressed in inclusionbodies, and A-L-E2 and A-L-E2s were dissolved in 4M and 8M urea, whileother fusion proteins were only dissolved in 8M urea. In addition, theresults also showed that after dialysis and renaturation, the fusionproteins of a purity of about 80% were obtained.

FIG. 3 shows the SDS-PAGE result of the fusion protein A-L-E2 purifiedby chromatography, wherein Lane 1 refers to A-L-E2 which is renatured toPBS after purification by chromatography, Lane 2 refers to a A-L-E2sample of Lane 1 boiled in boiling water for 10 mins. The results showedthat after two-step chromatography, A-L-E2 could reach a purity of above90%.

FIG. 4 shows the results of Western blotting using the fusion proteinsconstructed in Example 2 and HEV neutralizing monoclonal antibody 8C11.Lane M: protein molecular weight marker; Lane 1: Control proteinHEV-239; Lane 2: Control protein E2, Lane 3: CRM197-L-E2; Lane 4:CRM197-L-E2s; Lane 5: 389-L-E2; Lane 6: 389-L-E2s; Lane 7: 389-E2s; Lane8: A-L-E2; Lane 9: A-L-E2s; Lane 10: A-E2s. The results showed that allthe fusion proteins tested had significant reactivity with theHEV-specific neutralizing monoclonal antibody 8C11.

FIGS. 5A-5B show the results of indirect ELISA using the fusion proteinsconstructed in Example 2 and HEV-specific monoclonal antibody. Theabscissa refers to HEV-specific monoclonal antibody or CRM197 polyclonalantiserum for ELISA, and the ordinate refers to OD value determined byELISA at the same antibody dilution. FIG. 5A shows the ELISA result ofthe fusion proteins comprising E2, and FIG. 5B shows the ELISA result ofthe fusion proteins comprising E2s. The results showed that thereactivity of E2s protein with HEV-specific monoclonal antibody wassignificantly enhanced, after fusion of E2s protein with CRM197 or afragment thereof, wherein the reactivity of A-L-E2s and A-E2S wasenhanced most significantly; the reactivity of E2 protein withHEV-specific monoclonal antibody was retained or enhanced, after fusionof E2 protein with CRM197 or a fragment thereof.

FIG. 6 shows the results of indirect ELISA using the proteins A-L-E2,HEV-239 or E2 and HEV-specific monoclonal antibody, wherein the cutoffvalue is defined as three times of the average negative value. Theresults showed that the reactivity of A-L-E2 with HEV-specificmonoclonal antibody is comparable to that of HEV-239 and E2.

FIG. 7 shows the analytic result of Sedimentation Velocity (SV) of thefusion protein A-L-E2. The result showed that the fusion protein A-L-E2was mainly present in a form of dimer, and tetramer is present in asmall amount.

FIGS. 8A-8B show the comparison of immunogenicity between the fusionproteins constructed in Example 2 and HEV-239. The primary immunizationwas performed at week 0, and booster immunization was performed at week2 and 4, wherein the dose for both the primary immunization and thebooster immunization was 5 μg or 0.5 μg. FIG. 8A shows the comparisonresult of the antibody titer of immune serum in 5 μg-dose groups, andFIG. 8B shows the comparison result of the antibody titer of immuneserum in 0.5 μg-dose groups. The results showed that seroconversionagainst HEV occurred in mice serum at week 4 in 5 μg- and 0.5 μg-dosegroups, and the antibody titer reached the highest value at week 5 or 6.In particular, in 5 μg-dose group, the highest antibody titer wasobtained when A-L-E2 was used, which reached 10⁶ at week 6; and theantibody titers induced by the fusion proteins were higher than orcomparable to that of HEV-239 protein. In 0.5 μg-dose groups, theantibody titers of the fusion proteins were significantly higher thanthat of HEV-239, and the antibody titer induced by A-L-E2 protein atweek 5 reached 10⁶. In addition, seroconversion did not occur in immuneserum when using E2 and E2s in 5 μg- and 0.5 μg-dose groups. As seenfrom the results above, the immunogenicity of the fusion proteinsconstructed in Example 2 were significantly higher than the antigenprotein (E2 and E2s) alone, indicating that the CRM197 of the inventionor a fragment thereof significantly enhanced immunogenicity of theantigen protein fused therewith, and could be used as intramolecularadjuvant.

FIG. 9 shows the clone design of the fusion proteins constructed inExample 6, wherein the linker used (Linker, also referred to L for shortin the present application) is a flexible fragment consisting of 10amino acid residues, whose sequence is GGGGSGGGGS (SEQ ID NO:57); theCRM197 used comprised 535 amino acids, whose sequence is set forth inSEQ ID NO: 2; 389 refers to a polypeptide comprising amino acid residuesfrom positions 1 to 389 (aa 1-389) of CRM197; A refers to a polypeptidecomprising amino acid residues from positions 1 to 190 (aa 1-190) ofCRM197; M2 refers to an influenza virus M2 protein, whose sequence isset forth in SEQ ID NO: 32; M2e refers to a polypeptide comprising aminoacid residues from positions 1 to 24 (aa 1-24) of the influenza virus M2protein.

FIGS. 10A-10F show the SDS-PAGE analytic results of expression,purification and renaturation of the fusion proteins constructed inExample 6, wherein Lane M: protein molecular weight marker.

FIG. 10A used the samples that were the precipitate (i.e. inclusionbody) and the supernatant obtained by centrifuging the disruptedbacteria after ultrasonication:

Lane 1: the inclusion body obtained from the bacteria transformed withCRM197-L-M2e;

Lane 2: the supernatant obtained from the bacteria transformed withCRM197-L-M2e;

Lane 3: the inclusion body obtained from the bacteria transformed with389-L-M2e;

Lane 4: the supernatant obtained from the bacteria transformed with389-L-M2e; Lane 5: the inclusion body obtained from the bacteriatransformed with A-L-M2e; Lane 6: the supernatant obtained from thebacteria transformed with A-L-M2e.

FIG. 10B used the samples that were the precipitate (i.e. inclusionbody) and the supernatant obtained by centrifuging the disruptedbacteria after ultrasonication:

Lane 1: the inclusion body obtained from the bacteria transformed withM2e-L-A;

Lane 2: the supernatant obtained from the bacteria transformed withM2e-L-A; Lane 3: the inclusion body obtained from the bacteriatransformed with M2e-L-389;

Lane 4: the supernatant obtained from the bacteria transformed withM2e-L-389; Lane 5: the inclusion body obtained from the bacteriatransformed with M2e-L-CRM197;

Lane 6: the supernatant obtained from the bacteria transformed withM2e-L-CRM197.

FIG. 10C used the samples that were the fusion proteins isolated andrenatured into PBS, wherein no β-mercaptoethanol was used duringSDS-PAGE analysis, and the protein samples were treated by boiling (for10 min) or not:

Lane 1: A-L-M2e protein, not treated by boiling;

Lane 2: A-L-M2e protein, treated by boiling;

Lane 3: 389-L-M2e protein, not treated by boiling;

Lane 4: 389-L-M2e protein, treated by boiling;

Lane 5: CRM197-L-M2e protein, not treated by boiling;

Lane 6: CRM197-L-M2e protein, treated by boiling.

FIG. 10D used the samples that were the fusion proteins isolated andrenatured into PBS, wherein β-mercaptoethanol was used during SDS-PAGEanalysis, and the protein samples were treated by boiling (for 10 min)or not:

Lane 1: A-L-M2e protein, not treated by boiling;

Lane 2: A-L-M2e protein, treated by boiling;

Lane 3: 389-L-M2e protein, not treated by boiling;

Lane 4: 389-L-M2e protein, treated by boiling;

Lane 5: CRM197-L-M2e protein, not treated by boiling;

Lane 6: CRM197-L-M2e protein, treated by boiling.

FIG. 10E used the samples that were the fusion proteins isolated andrenatured into PBS, wherein no β-mercaptoethanol was used duringSDS-PAGE analysis, and the protein samples were treated by boiling (for10 min) or not:

Lane 1: M2e-L-A protein, not treated by boiling;

Lane 2: M2e-L-A protein, treated by boiling;

Lane 3: M2e-L-389 protein, not treated by boiling;

Lane 4: M2e-L-389 protein, treated by boiling;

Lane 5: M2e-L-CRM197 protein, not treated by boiling;

Lane 6: M2e-L-CRM197 protein, treated by boiling.

FIG. 10F used the samples that were the fusion proteins isolated andrenatured into PBS, wherein β-mercaptoethanol was used during SDS-PAGEanalysis, and the protein samples were treated by boiling (for 10 min)or not:

Lane 1: M2e-L-A protein, not treated by boiling;

Lane 2: M2e-L-A protein, treated by boiling;

Lane 3: M2e-L-389 protein, not treated by boiling;

Lane 4: M2e-L-389 protein, treated by boiling;

Lane 5: M2e-L-CRM197 protein, not treated by boiling;

Lane 6: M2e-L-CRM197 protein, treated by boiling.

The results shown in FIGS. 10A-10F indicated that all the constructedfusion proteins could be expressed in inclusion bodies, and afterpurification and renaturation, the fusion proteins with a purity ofabout 80% could be obtained.

FIGS. 11A-11H show the results of Western blotting using the fusionproteins constructed in Example 6 and anti-M2e monoclonal antibody 5D1and CRM197 monoclonal antibody 1E6. The samples represented by Lanes 1-6in FIGS. 11A, 11B, 11C and 11D correspond to the samples represented byLanes 1-6 in FIGS. 10C, 10D, 10E and 10F, respectively, wherein anti-M2especific monoclonal antibody 5D1 was used. The samples represented byLanes 1-6 in FIGS. 11E, 11F, 11G and 11H correspond to the samplesrepresented by Lanes 1-6 in FIGS. 10C, 10D, 10E and 10F, respectively,wherein CRM197 specific monoclonal antibody 1E6 was used. The resultsshowed that all the tested fusion proteins had significant reactivitywith anti-M2e specific monoclonal antibody 5D1 and CRM197 specificmonoclonal antibody 1E6.

FIGS. 12A-12B show the results of indirect ELISA using the fusionproteins constructed in Example 6 and various anti-M2e specificmonoclonal antibodies. The abscissa refers to anti-M2e specificmonoclonal antibodies and anti-CRM197 specific monoclonal antibodies forELISA, and the ordinate refers to OD value determined by ELISA at thesame antibody dilution. FIG. 12A shows the ELISA result of the fusionprotein in which M2e was fused to the C-terminus of CRM197 or a fragmentthereof, and FIG. 12B shows the ELISA result of the fusion protein inwhich M2e was fused to the N-terminus of CRM197 or a fragment thereof.The results showed that the fusion protein comprising M2e protein andCRM197 or a fragment thereof retained or enhanced the reactivity withvarious anti-M2e specific monoclonal antibodies, as compared to M2eprotein alone.

FIGS. 13A-13F show the analytic results of Sedimentation Velocity (SV)of the fusion proteins constructed in Example 6, wherein FIG. 13A:CRM197-L-M2e; FIG. 13B: 389-L-M2e; FIG. 13C: A-L-M2e; FIG. 13D:M2e-L-CRM197; FIG. 13E: M2e-L-389; FIG. 13F: M2e-L-A. The results showedthat the fusion proteins A-L-M2e and M2e-L-A were mainly present in aform of monomer and tetramer; and 389-L-M2e was mainly present in a formof dimer and polymer; M2e-L-389 was mainly present in a form of monomerand polymer; CRM197-L-M2e was mainly present in a form of dimer andpolymer; and M2e-L-CRM197 was mainly present in a form of monomer andpolymer.

FIGS. 14A-14B show the comparison of immunogenicity between the fusionproteins constructed in Example 6 and GST-M2e. The primary immunizationwas performed at week 0, and booster immunization was performed at week2 and 4, wherein the dose for both the primary immunization and thebooster immunization was 5 μg or 0.5 μs. FIG. 14A shows the comparisonresult of the antibody titer of immune serum in 5 μg-dose groups, andFIG. 14B shows the comparison result of the antibody titer of immuneserum in 0.5 μg-dose groups. The results showed that after the secondbooster immunization, the antibody titers induced by the fusion proteinswere significantly higher than GST-M2e alone in 5 μg- and 0.5 μg-dosegroups. As seen from the results above, the immunogenicity of the fusionproteins constructed in Example 6 were significantly higher than theantigen protein (GST-M2e) alone, indicating that the CRM197 of theinvention or a fragment thereof (no matter located at N-terminus orC-terminus of the fusion protein) significantly enhanced immunogenicityof the antigen protein fused therewith, and could be used asintramolecular adjuvant.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

The present invention is illustrated by reference to the followingexamples (which are used only for the purpose of illustrating thepresent invention and are not intended to limit the protection scope ofthe present invention).

Unless indicated otherwise, the molecular biological experimentalmethods and immunological assays used in the present invention arecarried out substantially in accordance with the methods as described inSambrook J et al., Molecular Cloning: A Laboratory Manual (SecondEdition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubelet al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley &Sons, Inc., 1995; restriction endonucleases are used under theconditions recommended by manufacturers of the products. The reagentsused in the present invention, whose manufacturers are not clearlyindicated, are conventional products in the art or commerciallyavailable. Those skilled in the art understand that the examples areused for illustrating the present invention, but not intended to limitthe protection scope of the present invention.

Example 1. Clone of CRM197 Gene

Genomic DNA extracted from Diphtheria bacillus C7 (β197) strain obtainedfrom ATCC (NO 53281) was used as template for the PCR reaction, whereinthe forward primer was CRM197F (SEQ ID NO: 19), and the reverse primerwas CRM197R (SEQ ID NO: 20). The PCR reaction was performed in a PCRapparatus (Biometra T3) under the following conditions, to prepare thefull-length gene encoding CRM197.

94° C. denaturation 10 min 1 cycle 94° C. denaturation 1.5 min 20cycles  58° C. annealing 1.5 min 72° C. elongation 1.5 min 72° C.elongation 10 min 1 cycle

After PCR amplification, a product of about 1.6 kb in length, wasobtained. After sequencing, the nucleotide sequence (SEQ ID NO: 1) ofthe amplification product (i.e. the full-length gene of CRM197) wasobtained, and the amino acid sequence encoded thereby was set forth inSEQ ID NO: 2.

Example 2. Design and Clone of Fusion Proteins Comprising CRM197 or aFragment Thereof and an HEV Capsid Protein Fragment

In the Example, vectors expressing the fusion proteins were constructedexemplarily. The clone design of various exemplary fusion proteinsconstructed is shown in FIG. 1, wherein the fusion proteins eachcomprise CRM197 or a fragment thereof and an HEV capsid proteinfragment, optionally using a linker.

Clone of Fusion Proteins Comprising a Linker

The amplification product (i.e. the full-length gene of CRM197) obtainedin the Example 1 was used as template. The forward primer was CRM197F(SEQ ID NO: 19), at the 5′ terminal of which the restrictionendonuclease NdeI site CAT ATG was introduced, wherein ATG was theinitiation codon in E. coli system. The reverse primers wereCRM197-linker R (SEQ ID NO: 21), 389-linker R (SEQ ID NO: 22), andA-linker R (SEQ ID NO: 23), respectively, at the 5′ terminal of whichthe restriction endonuclease BamHI site GGA TCC was introduced. The PCRreaction was performed in a PCR thermocycler (Biometra T3) under thefollowing conditions. The sequences of the primers used were shown inTable 1.

94° C. denaturation 10 min 1 cycle 94° C. denaturation 1.5 min 20 cycle 58° C. annealing 1.5 min 72° C. elongation 1.5 min 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 1600 bp, 1200 bpand 600 bp in length, respectively.

In addition, pTO-T7-E2 (Li, et al. JBC. 2005. 28(5): 3400-3406) was usedas template. The forward primers were E2F (SEQ ID NO: 24) and E2sF (SEQID NO: 25), respectively, at the 5′ terminal of which the restrictionendonuclease BamHI site GGA TCC was introduced. The reverse primer wasDrp59R (SEQ ID NO: 26), at the 5′ terminal of which the restrictionendonuclease EcoRI site GAA TTC was introduced. The PCR reaction wasperformed in a PCR thermocycler (Biometra T3) under the followingconditions.

94° C. denaturation 10 min 1 cycle 94° C. denaturation 50 sec 20 cycle 58° C. annealing 50 sec 72° C. elongation 50 sec 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 600 bp and 450 bpin length, respectively.

The amplification products as obtained above were linked intocommercially available pMD 18-T vector (produced by TAKARA Co.),respectively, and designated as pMD 18-T-CRM197-L, pMD 18-T-389-L andpMD 18-T-A-L as well as pMD 18-T-E2 and pMD 18-T-E2s. As identified byNdeI/BamHI and BamHI/EcoRI enzyme cleavage, respectively, the positiveclones pMD 18-T-CRM197-L, pMD 18-T-389-L, pMD 18-T-A-L, pMD 18-T-E2 andpMD 18-T-E2s were obtained.

As confirmed by M13(+) primer, correct nucleotide sequences of interestwere inserted into the obtained pMD 18-T-CRM197-L, pMD 18-T-389-L, pMD18-T-A-L, pMD 18-T-E2 and pMD 18-T-E2s, respectively.

The plasmids pMD 18-T-CRM197-L, pMD 18-T-389-L and pMD 18-T-A-L weredigested by NdeI/BamHI enzyme. The fragments obtained by enzyme cleavagewere linked into the prokaryotic expression vector pTO-T7 digested byNdeI/BamHI enzyme (Luo Wenxin et al., Chinese Journal of Biotechnology,2000, 16:53-57), and were transformed into E. coli ER2566 (purchasedfrom Invitrogen Co.); after extraction of plasmids, as identified byNdeI/BamHI enzyme cleavage, the positive plasmids pTO-T7-CRM197-L,pTO-T7-389-L and pTO-T7-A-L, into which CRM197-L, 389-L and A-L wereinserted, respectively, were obtained.

pTO-T7-CRM197-L, pTO-T7-389-L, pTO-T7-A-L, pMD 18-T-E2 and pMD 18-T-E2swere digested by BamHI/EcoRI enzyme. Each of the obtained E2 and E2sfragments was linked into the vectors pTO-T7-CRM197-L, pTO-T7-389-L andpTO-T7-A-L digested by BamHI/EcoRI enzyme, respectively. As identifiedby NdeI/EcoRI enzyme cleavage, the positive expression vectorspTO-T7-CRM197-L-E2, pTO-T7-CRM197-L-E2s, pTO-T7-389-L-E2,pTO-T7-389-L-E2s, pTO-T7-A-L-E2 and pTO-T7-A-L-E2s, into whichCRM197-L-E2 (SEQ ID NO:3, 4), CRM197-L-E2s (SEQ ID NO:5, 6), 389-L-E2(SEQ ID NO:7, 8), 389-L-E2s (SEQ ID NO:9, 10), A-L-E2 (SEQ ID NO:11, 12)or A-L-E2s (SEQ ID NO:13, 14) was inserted, respectively, were obtained.

Clone of the Fusion Proteins 389-E2s and A-E2s without a Linker

The vectors expressing 389-E2s and A-E2s were constructed by three PCRreactions. For the first PCR reaction, the full-length gene of CRM197was used as template. The forward primer was CRM197F, at the 5′ terminalof which the restriction endonuclease NdeI site CAT ATG was introduced,wherein ATG was the initiation codon in E. coli system. The reverseprimers were 389-E2s R (SEQ ID NO: 27) and A-E2s R (SEQ ID NO: 28),respectively. The amplification was performed to obtain the N-terminalfragments of the fusion proteins. For the second PCR reaction, thefull-length gene of CRM197 was used as template. The forward primer were389-E2s F (SEQ ID NO: 29) and A-E2s F (SEQ ID NO:30), respectively. Thereverse primer was DrP59 R, at the 5′ terminal of which the restrictionendonuclease EcoRI site GAA TTC was introduced. The amplification wasperformed to obtain the C-terminal fragments of the fusion proteins. Thefirst and second PCR reactions were performed in a PCR thermocycler(Biometra T3) under the following conditions.

94° C. denaturation 10 min 1 cycle 94° C. denaturation 50 sec 20 cycle 58° C. annealing 50 sec 72° C. elongation 50 sec 72° C. elongation 10min 1 cycle

For the third PCR reaction, the amplification products of the first andsecond PCR reactions were used as templates (for example, the twofragments obtained by using 389-E2sF and 389-E2sR as primers were usedas template for amplification of 389-E2s), and CRM197F and DrP59R wereused as primers. The amplification was performed in a PCR thermocycler(Biometra T3) under the following conditions.

94° C. denaturation 10 min 1 cycle 94° C. denaturation 50 sec 20 cycle 58° C. annealing 50 sec 72° C. elongation 50 sec 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 1600 bp and 1000bp in length, respectively. The amplification products obtained abovewere linked into commercially available pMD 18-T vector (produced byTAKARA Co.), respectively. As identified by NdeI/EcoRI enzyme cleavage,the positive clones pMD 18-T-389-E2s and pMD 18-T-A-E2s were obtained.

As confirmed by M13(+) primer, correct nucleotide sequences of SEQ IDNO:15 and SEQ ID NO:17 (which encoded the amino acid sequences of SEQ IDNO:16 and SEQ ID NO:18, respectively) were inserted into the obtainedpMD 18-T-389-E2s and pMD 18-T-A-E2s, respectively.

The plasmids pMD 18-T-389-E2s and pMD 18-T-A-E2s were digested byNdeI/EcoRI enzyme. The fragments obtained by enzyme cleavage were thenlinked into the prokaryotic expression vector pTO-T7 digested byNdeI/EcoRI enzyme (Luo Wenxin et al., Chinese Journal of Biotechnology,2000, 16:53-57). As identified by NdeI/EcoRI enzyme cleavage, thepositive plasmids pTO-T7-389-E2s and pTO-T7-A-E2s, into which 389-E2sand A-E2s were inserted, respectively, were obtained.

The sequences of the primers used in the Example were shown in Table 1.

TABLE 1  Primer sequences SEQ ID NO: Primer Name Primer sequence (5′-3′)19 CRM197F CATATGGGCGCTGATGATGTTGTTGATTCTTCT 20 CRM197RGAATTCCCCACTACCTTTCAGCTTTTG 21 CRM197-GGATCCACCGCCACCGCTGCCACCGCCACCGCTG linker R CCACCGCCACCGCTTTTGAT 22 389-GGATCCACCGCCACCGCTGCCACCGCCACCGCTG linker R CCACCGCCACCAAATGGTTGC 23A-linker R GGATCCACCGCCACCGCTGCCACCGCCACCGCTG CCACCGCCACCACGATTTCCTGCAC24 E2F GGATCCCAGCTGTTCTACTCTCGTC 25 E2sF GGATCCTCCCCAGCCCCATCGCGC 26Drp59R GAATTCCTAGCGCGGAGGGGGGGCT 27 389-E2s R GATGGGGCTGGGGAAAATGGTTG 28A-E2s R GATGGGGCTGGGGAACGATTTCCTGCAC 29 389-E2s F CGCAACCATTTTCCCCAGCCC30 A-E2s F GAAATCGTTCCCCAGCCCCAT

1 μL of plasmids pTO-T7-CRM197-L-E2, pTO-T7-CRM197-L-E2s,pTO-T7-389-L-E2, pTO-T7-389-L-E2s, pTO-T7-389-E2s, pTO-T7-A-L-E2,pTO-T7-A-L-E2s and pTO-T7-A-E2s (0.15 mg/ml) were separately used totransform 40 μL competent E. coli ER2566 (purchased from Invitrogen)prepared by the Calcium chloride method, and then the bacteria wereplated on solid LB medium (the components of the LB medium: 10 g/Lpeptone, 5 g/L yeast powder, and 10 g/L NaCl, the same below) containingkanamycin (at a final concentration of 100 mg/ml, the same below). Theplates were statically incubated at 37° C. for about 10-12 h untilindividual colonies could be observed clearly. Individual colonies fromthe plates were transferred to a tube containing 4 ml liquid LB mediumcontaining kanamycin. The cultures were incubated in a shaking incubatorat 180 rpm for 10 h at 37° C., and then 1 ml bacterial solutions wastaken and stored at −70° C.

Example 3. The Expression and Purification of the Fusion ProteinsConstructed in Example 2

Expression of Fusion Proteins and Purification of Inclusion Bodies

5 μL bacterial solution, taken from an ultra low temperature freezer at−70° C., was seeded to 5 mL liquid LB medium containing kanamycin, andthen was cultured at 37° C., 180 rpm under shaking until OD600 reachedabout 0.5. The resultant solution was transferred to 500 ml LB mediumcontaining kanamycin, and then was cultured at 37° C., 180 rpm undershaking for 4-5 h. When OD600 reached about 1.5, IPTG was added to afinal concentration of 0.4 mM, and the bacteria were induced undershaking at 37° C. for 4 h.

After induction, centrifugation was performed at 8000 g for 5 min tocollect the bacteria, and then the bacteria were re-suspended in a lysissolution at a ratio of 1 g bacteria to 10 mL lysis solution (20 mM Trisbuffer pH7.2, 300 mM NaCl), in ice-bath. The bacteria were treated witha sonicator (Sonics VCX750 Type Sonicator) (conditions: operating time15 min, pulse 2s, intermission 4s, output power 55%). The bacteriallysate was centrifuged at 12000 rpm, 4° C. for 5 min (the same below),the supernatant was discarded and the precipitate (i.e. inclusion body)was kept; 2% Triton-100 of the same volume was used for washing, theresult mixture was under vibration for 30 min, centrifuged, and thesupernatant was discarded. The precipitate was re-suspended in Buffer I(20 mM Tris-HCl pH8.0, 100 mM NaCl, 5 mM EDTA), under vibration for 30min, centrifuged, and the supernatant was discarded. The precipitate wasthen re-suspended in 2M urea, under vibration at 37° C. for 30 min,centrifuged, the supernatant and the precipitate were obtained. Thesupernatant was kept; and the precipitate was re-suspended in 4M urea inthe same volume, under vibration at 37° C. for 30 min, and centrifugedat 12000 rpm, 4° C. for 15 min to obtain the supernatant andprecipitate. The supernatant (i.e. the 4M urea-dissolved supernatant)was kept; and the precipitate was further in re-suspended in 8M urea inthe same volume, under vibration at 37° C. for 30 min, and centrifuged,and the supernatant (i.e. the 8M urea-dissolved supernatant) was kept.

The SDS-PAGE analytic results of the obtained fractions (Coomassiebrilliant blue staining was used for visualization, the same below, seethe methods in The Molecular Cloning Experiment Guide, 2^(nd) edition)was showed in FIG. 2. The results showed that the fusion proteins wereexpressed in inclusion bodies (see FIG. 2A), and CRM197-L-E2, 389-L-E2,A-L-E2, and A-E2s were mainly dissolved in 4M urea (see FIG. 2B),CRM197-L-E2s, 389-L-E2s, A-L-E2s, and 389-E2s were mainly dissolved in8M urea (see FIG. 2C). The 4M urea-dissolved supernatants or the 8Murea-dissolved supernatants containing the fusion protein, were dialyzedto PBS, respectively, to get the fusion proteins with a purity of about80% (see FIG. 2D).

Purification of the Fusion Protein A-L-E2 by Anion ExchangeChromatography

Sample: a solution of A-L-E2 protein with a purity of about 80% asobtained above.

Equipment: AKTA Explorer 100 preparative liquid chromatography systemproduced by GE Healthcare (i.e. the original Amersham Pharmacia Co.)

Chromatographic media: Q SEPHAROSE® Fast Flow (GE Healthcare Co.)

Column Volume: 15 mm×20 cm

Buffer: 20 mM phosphate buffer pH 7.7+4M urea

20 mM phosphate buffer pH 7.7+4M urea+1M NaCl

Flow Rate: 6 mL/min

Detector Wavelength: 280 nm

Elution protocol: eluting the protein of interest with 150 mM NaCl,eluting the undesired protein with 300 mM NaCl, and collecting thefraction eluted with 150 mM NaCl.

Purification of the Fusion Protein A-L-E2 by Hydrophobic InteractionChromatography

Equipment: AKTA Explorer 100 preparative liquid chromatography systemproduced by GE Healthcare (i.e. the original Amersham Pharmacia Co.)

Chromatographic media: Phenyl SEPHAROSE® Fast Flow (GE Healthcare Co.)

Column Volume: 15 mm×20 cm

Buffer: 20 mM phosphate buffer pH 7.7+4M urea+0.5 M (NH₄)₂SO₄

20 mM phosphate buffer pH 7.7+4M

Flow Rate: 5 mL/min

Detector Wavelength: 280 nm

Sample: the fraction eluted with 150 mM NaCl as obtained in the previousstep was dialyzed to a buffer (20 mM phosphate buffer pH 7.7+4M urea+0.5M (NH₄)₂SO₄), and then was used as sample.

Elution protocol: eluting the undesired protein with 0.3M (NH₄)₂SO₄,eluting the protein of interest with 0.1M and 0M (NH₄)₂SO₄, andcollecting the fraction eluted with 0.1M and 0M (NH₄)₂SO₄.

The fraction eluted with 0.1M and 0M (NH₄)₂SO₄ was dialyzed andrenatured into PBS, and then 10 μl was taken for SDS-PAGE analysis, andelectrophoresis bands were visualized by Coomassie brilliant bluestaining. The results showed that after the above purification steps,the fusion protein A-L-E2 had a purity of above 90% (See FIG. 3).

Example 4. Analysis of Properties of the Fusion Proteins Constructed inExample 2

Determination of the Reactivity of the Fusion Proteins with Antibodiesby Western Blotting

The reactivity of the fusion proteins with HEV neutralizing monoclonalantibody 8C11 (see, Zhang et al., Vaccine. 23(22): 2881-2892 (2005)) andanti-CRM197 polyclonal antiserum (which was prepared by immunizing micewith CRM197 through methods well known in the art, and the reactivity ofthe serum was confirmed by commercially available CRM197) weredetermined by Western blotting. The dialyzed and renatured samples weretransferred to nitrocellulose membrane for blotting after SDS-PAGEseparation; 5% skimmed milk was used to block the membrane for 2 h,monoclonal antibody 8C11 diluted at a certain ratio was then added(monoclonal antibody was diluted at 1:500, and polyclonal antiserum wasdiluted at 1:1000), and the reaction was carried out for 1 h. Themembrane was washed with TNT (50 mmol/L Tris.Cl (pH 7.5), 150 mmol/LNaCl, 0.05% Tween 20) for three times, 10 min for each time. GoatAnti-mouse alkaline phosphatase (KPL product) was then added, thereaction was carried out for 1 h, and the membrane was then washed withTNT for three times, 10 min for each time. NBT and BCIP (PROTOS product)were used for visualization. The results, as determined by Westernblotting using the fusion proteins and HEV neutralizing monoclonalantibody 8C11, were shown in FIG. 4. The results showed that all thetested fusion proteins had significant reactivity with HEV neutralizingmonoclonal antibody 8C11.

Determination of the Reactivity of the Fusion Proteins with Various HEVSpecific Antibodies by ELISA

The reactivity of the fusion proteins and the control proteins E2 andHEV-239 with various HEV specific antibodies (Gu Ying et al., ChineseJournal of Virology, 19(3): 217-223(2003)) was determined by indirectELISA. The dialyzed and renatured samples were diluted in 1×PBS (1μg/ml), and then were added to 96-well microplate (Beijing Wantai Co.)at 100 μl/well and incubated at 37° C. for 2 h. The coating solution wasdiscarded, the plate was washed with PBST (PBS+0.05% Tween-20) once, andthen the blocking solution (2% gelatin, 5‰ Casein, 1‰ PROCLIN® 300, inPBS) was added at 200 μl/well and incubated at 37° C. for 2 h. Theblocking solution was discarded when the detection was performed, andthe HEV monoclonal antibodies diluted at a certain ratio (when E2s andits fusion protein were detected, they were diluted at 1:10000; when E2and its fusion protein were detected, they were diluted at 1:100000;when the reactivity of A-L-E2, 239 and E2 proteins was compared, themonoclonal antibodies were subjected to 10-fold serial dilution wherein1 mg/ml was used as the initial concentration, and the polyclonalantibody at its initial concentration was subjected to dilution in thesame manner) was added at 100 μl/well. The mixture was incubated at 37°C. for 1-2 h. The plate was then washed with PBST for five times, andHRP-labeled Goat anti Mouse (KPL product) (1:5000) was then added at 100μl/well and was incubated at 37° C. for 30 min; the plate was thenwashed with PB ST for five times, HRP substrate (Beijing Wantai Co.) wasthen added at 100 μl/well and was incubated at 37° C. for 15 min. 2Msulphuric acid was added at 50 μl/well to stop the reaction, andMicroplate reader (Sunrise Type, product from Tecan Co.) was then usedto read OD450/620 value. The results of the ELISA using the fusionproteins with the monoclonal antibodies were shown in FIG. 5. Theresults showed that the reactivity of E2s protein with the monoclonalantibody was significantly enhanced, after its fusion with CRM197 or afragment thereof, wherein the reactivity of A-L-E2s and A-E2s wasenhanced most significantly; the reactivity of E2 protein withHEV-specific monoclonal antibody was retained or enhanced, after itsfusion with CRM197 or a fragment thereof.

Analysis of the Reactivity of the Fusion Protein A-L-E2 Purified byChromatography

The reactivity of the fusion protein A-L-E2, purified by two-stepchromatography, was analyzed by indirect ELISA (see the concrete processin the previous step). The ELISA result was shown in FIG. 6. The resultshowed that the reactivity of A-L-E2 with HEV specific monoclonalantibody was comparable to that of the control proteins HEV-239 and E2.

Analysis of Sedimentation Velocity (SV) of the Fusion Protein A-L-E2

The apparatus used in the experiment was US Beckman XL-A analyticsupercentrifuge, which was equipped with an optical detection system andAn-50Ti and An-60Ti rotators. The Sedimentation Velocity (SV) method(c(s) algorithm, see P. Schuck et al., Biophys J 78: 1606-1619(2000))was used to analyze the sedimentation coefficient of the fusion proteinA-L-E2. The analytic result was shown in FIG. 7. The result showed thatthe fusion protein A-L-E2 was mainly present in a form of dimer, andsome dimers might be further polymerized to form a tetramer.

Example 5. Analysis of Immunogenicity of the Fusion Proteins Constructedin Example 2

Antibody Titers Induced by the Fusion Proteins

The mice used in the experiment were female, 6-week old BALB/C mice. Byusing aluminum adjuvant, mice were immunized by intraperitonealinjection of the fusions proteins which were prepared by the methods inthe Example 3 and renatured to PBS and the control proteins HEV-239, E2and E2s, respectively. The injection volume was 1 ml, and two dosegroups (a 5 μg-dose group or a 0.5 μg-dose group) were used. The primaryimmunization was performed at week 0, and booster immunization wasperformed at week 2 and 4.

HEV-239 was used to coat a plate, and the antibody titers in serum asinduced by the fusion proteins and the control proteins, were measuredby similar indirect ELISA assay as described above. The detectionresults of the serum antibody titers within 3 months after immunizationwere shown in FIG. 8. The results showed that seroconversion occurred inmice serum at week 4 in both 5 μg- and 0.5 μg-dose groups, and theantibody titers reached the highest value at week 5 or 6. In particular,in 5 μg-dose group, the highest antibody titer was obtained when A-L-E2was used, which reached 10⁶ at week 6; and the antibody titers inducedby the fusion proteins were higher or comparable to that of HEV-239protein. In 0.5 μg-dose groups, the antibody titers of the fusionproteins were significantly higher than that of HEV-239, and theantibody titer induced by A-L-E2 protein reached 10⁶ at week 5. Inaddition, seroconversion did not occur in immune serum when using E2 andE2s, in 5 μg- and 0.5 μg-dose groups. As seen from the above results,the immunogenicity of the constructed fusion proteins were significantlyhigher than the antigen protein (E2 and E2s) alone, indicating that theCRM197 of the invention or a fragment thereof significantly enhancedimmunogenicity of the antigen protein fused therewith, and could be usedas intramolecular adjuvant.

Investigation on Median Effective Dose (ED50) of the Fusion ProteinA-L-E2

In the experiment, immunogenicity of fusion proteins was investigated bydetermining median effective dose (ED50). The experimental animals usedwere 3-4 week old female BALB/c mice. A-L-E2 was mixed with aluminumadjuvant, and the initial dose was 1 μg/mouse, and was subjected toserial dilution at 1:3, resulting in 8 dose groups in total. Inaddition, HEV-239 (HEV recombinant vaccine) was used as control, and theinitial dose was 1.6 μg/mouse, and was subjected to serial dilution at1:4, resulting in 4 dose groups in total. 6 mice were used in eachgroup. The immunization was carried out by single intraperitonealinjection.

Peripheral venous blood was taken after 4 weeks following immunization,serum was separated, and serological conversion rate was determined byELISA assay as described above. When the ELISA value of 100-fold dilutedserum was higher than the cutoff value (i.e. three times of the averagenegative value), the serum was regarded as positive. The medianeffective dose (ED50) was calculated by Reed-Muench method. Theserological conversion rate of the fusion protein A-L-E2 was shown inTable 2, and the serological conversion rate of HEV-239 vaccine wasshown in Table 3.

TABLE 2 ED50 of A-L-E2 for inducing seroconversion of HEV antibody inmice Number of Serological Number of mice with conversion Dose (μg) miceseroconversion rate ED50(μg) 1.0000 6 6 100% 0.0064 0.3333 6 6 100%0.1111 6 6 100% 0.0370 6 6 100% 0.0123 6 6 100% 0.0041 6 1 16.7%  0.00136 0  0% 0.0005 6 0  0%

TABLE 3 ED50 of HEV-239 vaccine for inducing seroconversion of HEVantibody in mice Number of Serological Number of mice with conversionDose (μg) mice seroconversion rate ED50(μg) 1.6 6 6  100% 0.071 0.4 6 583.3% 0.1 6 4 66.7% 0.025 6 0   0%

The results showed that ED50 of HEV-239 was 11 times of that of A-L-E2,indicating that CRM197 of the invention or a fragment thereofsignificantly enhanced immunogenicity of the antigen protein fusedtherewith, and could be used as intramolecular adjuvant. Meanwhile,since immunogenicity of the fusion protein A-L-E2 was significantlyhigher than that of HEV-239 vaccine in the form of virus like particle,the fusion protein might be used for the preparation of a new vaccinewhich is more effective for Hepatitis E.

Example 6. Design and Clone of Fusion Proteins Comprising CRM197 or aFragment Thereof and an Influenza Virus M2e Protein

In the Example, vectors expressing the fusion proteins were constructedexemplarily. The clone design of the exemplary fusion proteinsconstructed is shown in FIG. 9, wherein the fusion proteins eachcomprise CRM197 or a fragment thereof and an influenza virus M2eprotein, optionally using a linker.

Clone of Fusion Proteins

M2e Fused to the C-Terminus of CRM197 or a Fragment Thereof

The amplification product (i.e. the full-length gene of CRM197) obtainedin the Example 1 was used as template. The forward primer was CRM197F1(SEQ ID NO: 45), at the 5′ terminal of which the restrictionendonuclease NdeI site CAT ATG was introduced, wherein ATG was theinitiation codon in E. coli system. The reverse primers wereCRM197-linker R1 (SEQ ID NO: 46), 389-linker R1 (SEQ ID NO: 47) andA-linker R1 (SEQ ID NO: 48), respectively, at the 5′ terminal of whichthe restriction endonuclease BamHI site GGA TCC was introduced. The PCRreaction was performed in a PCR thermocycler (Biometra T3) under thefollowing conditions. The sequences of the primers used were shown inTable 4.

95° C. denaturation 10 min 1 cycle 95° C. denaturation 1.5 min 20 cycle 58° C. annealing 1.5 min 72° C. elongation 1.7 min 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 1600 bp, 1200 bpand 600 bp in length, respectively.

In addition, the plasmid PHW2000 (stored in our lab, comprising thefull-length gene of M2) was used as a template. The forward primer wasM2eF1 (SEQ ID NO: 49), at the 5′ terminal of which the restrictionendonuclease BamHI GGA TCC was introduced. The reverse primer was M2eR(SEQ ID NO: 50), at the 5′ terminal of which the restrictionendonuclease EcoRI site GAA TTC was introduced. The PCR reaction wasperformed in a PCR thermocycler (Biometra T3) under the followingconditions. The sequences of the primers used were shown in Table 4.

95° C. denaturation 10 min 1 cycle 95° C. denaturation 50 sec 20 cycle 58° C. annealing 50 sec 72° C. elongation 30 sec 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 70 bp in length,respectively.

The amplification products as obtained above were linked intocommercially available pMD 18-T vector (produced by TAKARA Co.),respectively, and designated as pMD 18-T-CRM197-L1, pMD 18-T-389-L1 andpMD 18-T-A-L1 as well as pMD 18-T-M2e. As identified by NdeI/BamHI andBamHI/EcoRI enzyme cleavage, respectively, the positive clones pMD18-T-CRM197-L1, pMD 18-T-389-L1, pMD 18-T-A-L1 and pMD 18-T-M2e wereobtained.

As confirmed by M13(+) primer, correct nucleotide sequences of interestwere inserted into the obtained plasmids pMD 18-T-CRM197-L1, pMD18-T-389-L1, pMD 18-T-A-L1 and pMD 18-T-M2e.

The plasmids pMD 18-T-CRM197-L1, pMD 18-T-389-L1 and pMD 18-T-A-L1 weredigested by NdeI/BamHI enzyme. The fragments obtained by enzyme cleavagewere linked into the prokaryotic expression vector pTO-T7 digested byNdeI/BamHI enzyme (Luo Wenxin et al., Chinese Journal of Biotechnology,2000, 16:53-57), and were transformed into E. coli ER2566 (purchasedfrom Invitrogen Co.); after extraction of plasmids, as identified byNdeI/BamHI enzyme cleavage, the positive plasmids pTO-T7-CRM197-L1,pTO-T7-389-L1 and pTO-T7-A-L1, into which the fragments CRM197-L1,389-L1 and A-L1 were inserted, respectively, were obtained.

pTO-T7-CRM197-L1, pTO-T7-389-L1, pTO-T7-A-L1 and pMD 18-T-M2e weredigested by BamHI/EcoRI enzyme. The obtained M2e fragment was linkedinto the vectors pTO-T7-CRM197-L1, pTO-T7-389-L1 and pTO-T7-A-L1digested by BamHI/EcoRI enzyme, respectively. As identified byNdeI/EcoRI enzyme cleavage, the positive expression vectorspTO-T7-CRM197-L-M2e, pTO-T7-389-L-M2e, and pTO-T7-A-L-M2e, into whichCRM197-L-M2e (SEQ ID NO:33, 34), 389-L-M2e (SEQ ID NO:35, 36), orA-L-M2e (SEQ ID NO:37, 38) was inserted respectively, were obtained.

M2e Fused to the N-Terminus of CRM197 or a Fragment Thereof.

The plasmid PHW2000 (stored in our lab, containing the full-length geneof M2) was used as template. The forward primer was M2eF2 (SEQ ID NO:51), at the 5′ terminal of which the restriction endonuclease NdeI CATATG was introduced, wherein ATG was the initiation codon in E. colisystem. The reverse primer was M2e-Linker R (SEQ ID NO: 52), at the 5′terminal of which the restriction endonuclease BamHI GGA TCC wasintroduced. The PCR reaction was performed in a PCR thermocycler(Biometra T3) under the following conditions.

95° C. denaturation 10 min 1 cycle 95° C. denaturation 50 sec 20 cycle 58° C. annealing 50 sec 72° C. elongation 30 sec 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 100 bp in length.

In addition, the amplification product (i.e. the full-length gene ofCRM197) obtained in the Example 1 was used as template. The forwardprimer was CRM197F2 (SEQ ID NO: 53), at the 5′ terminal of which therestriction endonuclease BamHI GGA TCC was introduced. The reverseprimers were CRM197 R2 (SEQ ID NO: 54), 389 R (SEQ ID NO: 55), and A R(SEQ ID NO: 56), at the 5′ terminal of which the restrictionendonuclease EcoRI site GAA TTC was introduced. The PCR reaction wasperformed in a PCR thermocycler (Biometra T3) under the followingconditions. The sequences of the primers used were shown in Table 4.

95° C. denaturation 10 min 1 cycle 95° C. denaturation 1.5 min 20 cycle 58° C. annealing 1.5 min 72° C. elongation 1.7 min 72° C. elongation 10min 1 cycle

The amplification products were DNA fragments of about 1600 bp, 1200 bpand 600 bp in length, respectively.

The amplification products as obtained above were linked intocommercially available pMD 18-T vector (produced by TAKARA Co.),respectively, and designated as pMD 18-T-M2e-L as well as pMD18-T-CRM197, pMD 18-T-389 and pMD 18-T-A, respectively. As identified byNdeI/BamHI and BamHI/EcoRI enzyme cleavage, respectively, the positiveclones pMD 18-T-CRM197, pMD 18-T-389, pMD 18-T-A, and pMD 18-T-M2e-Lwere obtained.

As confirmed by M13(+) primer, correct nucleotide sequences of interestwere inserted into the obtained plasmids pMD 18-T-CRM197, pMD 18-T-389,pMD 18-T-A, and pMD 18-T-M2e-L, respectively.

The plasmid pMD 18-T-M2e-L was digested by NdeI/BamHI enzyme. Thefragments obtained by enzyme cleavage were then linked into theprokaryotic expression vector pTO-T7 digested by NdeI/BamHI enzyme (LuoWenxin et al., Chinese Journal of Biotechnology, 2000, 16:53-57), andwas transformed into E. coli ER2566 (purchased from Invitrogen Co.);after extraction of plasmids, as identified by NdeI/BamHI enzymecleavage, the positive plasmid pTO-T7-M2e-L, into which the fragmentM2e-L was inserted, was obtained.

pTO-T7-M2e-L, pMD 18-T-CRM197, pMD 18-T-389 and pMD 18-T-A were digestedby BamHI/EcoRI enzyme. The obtained fragments CRM197, 389 and A werelinked into the vector pTO-T7-M2e-L digested by BamHI/EcoRI enzyme,respectively. As identified by NdeI/EcoRI enzyme cleavage, the positiveexpression vectors pTO-T7-M2e-L-CRM197, pTO-T7-M2e-L-389, andpTO-T7-M2e-L-A, into which M2e-L-CRM197 (SEQ ID NO:39, 40), M2e-L-389(SEQ ID NO:41, 42), and M2e-L-A (SEQ ID NO:43, 44) were insertedrespectively, were obtained.

The sequences of the primers used in the Example are listed in Table 4.

TABLE 4  Primer sequences SEQ ID Primer  NO: namesPrimer sequences (5′-3′) 45 CRM197 F1 CATATGGGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAA 46 CRM197- GGATCCGCTGCCACCGCCACCGCTGCCACCGCC linker R1ACCGCTTTTGAT 47 389-linker GGATCCGCTGCCACCGCCACCGCTGCCACCGCC R1ACCAAATGGTTG 48 A-linker  GGATCCGCTGCCACCGCCACCGCTGCCACCGCC R1ACCACGATTTCC 49 M2e F1 GGATCCATGAGTCTTCTAACCGAGGTCGAAACG CCT 50 M2e RGAATTCTTAATCACTTGAACCGTTGCATCTGCAC CCCCA 51 M2e F2CATATGATGAGTCTTCTAACCGAGGTCGAAACG CCT 52 M2e-LinkerGGATCCGCTGCCACCGCCACCGCTGCCACCGCC R ACCATCACTTGA 53 CRM197 F2GGATCCGGCGCTGATGATGTTGTTGATTCTTCTA AATCTTTTGTGATGGAA 54 CRM197 R2GAATTCTAAGCTTTTGATTTCAAAAAATAGCGAT AGCTTAGA 55 389RGAATTCTAAAAATGGTTGCGTTTTATGCCCCGGA GAATACGC 56 ARGAATTCTAAACGATTTCCTGCACAGGCTTGAGCC ATATACTC

1 μL of plasmids pTO-T7-CRM197-L-M2e, pTO-T7-389-L-M2e, pTO-T7-A-L-M2e,pTO-T7-M2e-L-CRM197, pTO-T7-M2e-L-389 and pTO-T7-M2e-L-A (0.15 mg/ml)were separately used to transform 40 μL competent E. coli ER2566(purchased from Invitrogen) prepared by the Calcium chloride method, andthen the bacteria were plated on solid LB medium (the components of theLB medium: 10 g/L peptone, 5 g/L yeast powder, and 10 g/L NaCl, the samebelow) containing kanamycin (at a final concentration of 100 mg/ml, thesame below). The plates were statically incubated at 37° C. for about10-12 h until individual colonies could be observed clearly. Individualcolonies from the plates were transferred to a tube containing 4 mlliquid LB medium containing kanamycin. The cultures were incubated in ashaking incubator at 180 rpm for 10 h at 37° C., and then 1 ml bacterialsolution was taken and stored at −70° C.

Example 7. The Expression, Isolation and Renaturation of the FusionProteins Constructed in Example 6

5 μL bacterial solution, taken from an ultra low temperature freezer at−70° C., was seeded to 5 mL liquid LB medium containing kanamycin, andthen was cultured at 37° C., 180 rpm under shaking until OD600 reachedabout 0.5. The resultant solution was transferred to 500 ml LB mediumcontaining kanamycin, and then was cultured at 37° C., 180 rpm undershaking for 4-5 h. When OD600 reached about 1.5, IPTG was added to afinal concentration of 0.4 mM, and the bacteria were induced undershaking at 37° C. for 4 h.

After induction, centrifugation was performed at 8000 g for 5 min tocollect the bacteria, and then the bacteria was re-suspended in a lysissolution at a ratio of 1 g bacteria to 10 mL lysis solution (20 mM Trisbuffer pH7.2, 300 mM NaCl), in ice-bath. The bacteria was treated with asonicator (Sonics VCX750 Type Sonicator) (conditions: operating time 15min, pulse 2s, intermission 4s, output power 55%). The bacterial lysatewas centrifuged at 12000 rpm, 4° C. for 5 min (the same below), and thesupernatant and the precipitate (i.e. inclusion body) after disruptingthe bacteria by ultrasonication were collected, respectively. 2%Triton-100 of the same volume was used for washing the precipitate, theresult mixture was under vibration for 30 min, centrifuged, and thesupernatant was discarded. The precipitate was re-suspended in Buffer I(20 mM Tris-HCl pH8.0, 100 mM NaCl, 5 mM EDTA), under vibration for 30min, centrifuged, and the supernatant was discarded. The precipitate wasthen re-suspended in 2M urea, under vibration at 37° C. for 30 min,centrifuged, and the supernatant and the precipitate were obtained. Thesupernatant was kept; and the precipitate was re-suspended in 4M urea inthe same volume, under vibration at 37° C. for 30 min, and centrifugedat 12000 rpm, 4° C. for 15 min to obtain the supernatant andprecipitate. The supernatant (i.e. the 4M urea-dissolved supernatant)was kept; and the precipitate was further in re-suspended in 8M urea inthe same volume, under vibration at 37° C. for 30 min, and centrifuged,and the supernatant (i.e. the 8M urea-dissolved supernatant) was kept.

The fractions obtained were analyzed by SDS-PAGE (Coomassie brilliantblue staining was used for visualization, the same below, see themethods in The Molecular Cloning Experiment Guide, 2^(nd) edition). Theresults showed that the fusion proteins were expressed in inclusionbodies (see FIGS. 10A and 10B), CRM197-L-M2e, 389-L-M2e, M2e-L-CRM197and M2e-L-389 were mainly dissolved in 8M urea, and A-L-M2e and M2e-L-Awere mainly dissolved in 4M urea. The 4M urea-dissolved supernatantscontaining A-L-M2e or M2e-L-A or the 8M urea-dissolved supernatantscontaining CRM197-L-M2e, 389-L-M2e, M2e-L-CRM197 or M2e-L-389, weredialyzed to PBS, respectively, to get the fusion proteins with a purityof about 80% (see FIGS. 10C-10F).

Example 8. Analysis of Properties of the Fusion Proteins Constructed inExample 6

Determination of the Reactivity of the Fusion Proteins with Antibodiesby Western Blotting

The reactivity of the fusion proteins with influenza virus M2emonoclonal antibody 5D1 and CRM197 monoclonal antibody 1E6 (prepared inthe laboratory) were determined by Western blotting. The dialyzed andrenatured samples were transferred to nitrocellulose membrane forblotting after SDS-PAGE separation; 5% skimmed milk was used to blockthe membrane for 2 h, and then the monoclonal antibody 5D1 diluted at1:500 was added. The reaction was carried out for 1 h. The membrane wasthen washed with TNT (50 mmol/L Tris.Cl (pH 7.5), 150 mmol/L NaCl, 0.05%Tween 20) for three times, 10 min for each time. Goat Anti-mousealkaline phosphatase (KPL product) was then added. The reaction wascarried out for 1 h, and the membrane was then washed with TNT for threetimes, 10 min for each time. NBT and BCIP (PROTOS product) were used forvisualization. The results, as determined by Western blotting using thefusion proteins and influenza virus M2e monoclonal antibody 5D1 (FIGS.11A-11D) or CRM197 monoclonal antibody 1E6 (FIGS. 11E-11H), were shownin FIG. 11. The results showed that all the tested fusion proteins hadsignificant reactivity with influenza virus M2e-specific monoclonalantibody 5D1 and CRM197 specific monoclonal antibody 1E6.

Determination of the Reactivity of the Fusion Proteins with Various M2eSpecific Monoclonal Antibodies and CRM197 Specific Antibody by ELISA

The reactivity of the fusion proteins and the control protein GST-M2ewith various M2e specific antibodies and CRM197 specific monoclonalantibody 1E6 (the antibodies used in the experiment were known in theprior art, or commercially available or prepared in the laboratory) wasdetermined by indirect ELISA. For example, 019 antibody is a protectiveantibody against influenza known in the prior art (see, Fu et al.,Virology, 2009, 385:218-226). The dialyzed and renatured samples werediluted in 1×PBS (1 μg/ml), and then were added to 96-well microplate(Beijing Wantai Co.) at 100 μl/well and incubated at 37° C. for 2 h. Thecoating solution was discarded, the plate was washed with PBST(PBS+0.05% TWEEN® 20) once, and then the blocking solution (2% gelatin,5‰ Casein, 1‰ PROCLIN® 300, in PBS) was added at 180 μl/well andincubated at 37° C. for 2 h. The blocking solution was discarded whenthe detection was performed, and the anti-M2e antibody or CRM197antibody diluted at a certain ratio (0.002 mg/ml was used as the initialconcentration for 2-fold gradient dilution) was added at 100 μl/well.The mixture was incubated at 37° C. for 1 h. The plate was washed withPBST for five times, HRP-labeled Goat anti Mouse (KPL product) (1:5000)was then added at 100 μl/well and was incubated at 37° C. for 30 min.The plate was washed with PBST for five times, HRP substrate (BeijingWantai Co.) was then added at 100 μl/well and was incubated at 37° C.for 15 min. 2M sulphuric acid was added at 50 μl/well to stop thereaction, and Microplate reader (Sunrise Type, product from Tecan Co.)was then used to read OD450/620 value. The results of the ELISA usingthe fusion proteins with the antibodies were shown in FIGS. 12A and 12B.The results showed that as compared to M2e protein alone, the reactivityof M2e protein with various anti-M2e specific monoclonal antibodies wasretained or enhanced after its fusion with CRM197 or a fragment thereof.

Analysis of Sedimentation Velocity (SV) of the Fusion Proteins

The apparatus used in the experiment was US Beckman XL-A analyticsupercentrifuge, which was equipped with an optical detection system andAn-50Ti and An-60Ti rotators. The Sedimentation Velocity (SV) method(c(s) algorithm, see P. Schuck et al., Biophys J 78: 1606-1619(2000))was used to analyze the sedimentation coefficient of the fusionproteins. The analytic results were shown in FIGS. 13A-13F. The resultsshowed that among the fusion proteins constructed in Example 6, A-L-M2eand M2e-L-A were mainly present in a form of monomer and tetramer; and389-L-M2e was mainly present in a form of dimer and polymer; M2e-L-389was mainly present in a form of monomer and polymer; CRM197-L-M2e wasmainly present in a form of dimer and polymer; and M2e-L-CRM197 wasmainly present in a form of monomer and polymer.

Example 9. Analysis of Immunogenicity of the Fusion Proteins Constructedin Example 6

The mice used in the experiment were female, 6-week old BALB/C mice. Byusing aluminum adjuvant, mice were immunized by intraperitonealinjection of the fusions proteins as constructed in Example 6 andrenatured to PBS and the control protein GST-M2e, respectively. Theinjection volume was 1 ml, and two dose groups (a 5 μg-dose group or a0.5 μg-dose group) were used. The primary immunization was performed atweek 0, and booster immunization was performed at week 2 and 4.

GST-M2e was used to coat a plate, and the antibody titers in serum asinduced by the fusion proteins and control protein, were measured bysimilar indirect ELISA assay as described above. The detection resultsof the serum antibody titers within 4 months after immunization wereshown in FIGS. 14A and 14B. The results showed that after the secondbooster immunization, immunogenicity of the constructed fusion proteinswas significantly higher than the antigen protein (GST-M2e) alone,indicating that the CRM197 of the invention or a fragment thereof (nomatter being located at the N-terminus or C-terminus of the fusionprotein) significantly enhanced immunogenicity of the antigen proteinfused therewith, and could be used as intramolecular adjuvant.

Although the specific embodiments of the invention have been describedin details, those skilled in the art would understand that, according tothe teachings disclosed in the specification, various modifications andchanges can be made without departing from the sprit or scope of theinvention as generally described, and that such modifications andchanges are within the scope of the present invention. The scope of thepresent invention is given by the appended claims and any equivalentsthereof.

The invention claimed is:
 1. A method for enhancing immunogenicity of atarget protein, comprising fusing the target protein to a fragment ofCRM197, optionally via a linker, to obtain a fusion protein, wherein thefragment of CRM197 consists of aa 1-190 or aa 1-389 of SEQ ID NO: 2, andwherein the fragment of CRM197 enhances immunogenicity of the targetprotein.
 2. The method of claim 1, wherein the fusion protein isobtained by fusion expression of the fragment of CRM197 with the targetprotein, optionally using a linker.
 3. The method of claim 1, whereinthe fusion protein comprises the fragment of CRM197 which is linked tothe N-terminus and/or C-terminus of the target protein, optionally via alinker.
 4. The method of claim 1, wherein the target protein is HEVcapsid protein or an immunogenic fragment thereof.
 5. The method ofclaim 4, wherein the immunogenic fragment of HEV capsid proteincomprises or is HEV-239 (aa 368-606 of HEV capsid protein), E2 (aa394-606 of HEV capsid protein) or E2s (aa 455-606 of HEV capsidprotein).
 6. The method of claim 4, wherein the fusion protein comprises(a) the fragment of CRM197, and (b) HEV capsid protein or theimmunogenic fragment of HEV capsid protein; wherein (a) and (b) arelinked together, optionally via a linker.
 7. The method of claim 4,wherein the fusion protein has an amino acid sequence as set forth inSEQ ID NO: 8, 10, 12, 14, 16 or
 18. 8. The method of claim 1, whereinthe target protein is influenza virus M2 protein or an immunogenicfragment thereof.
 9. The method of claim 8, wherein the immunogenicfragment of M2 protein comprises or is M2e (aa 1-24 of M2 protein). 10.The method of claim 8, wherein the fusion protein comprises (a) thefragment of CRM197, and (b) influenza virus M2 protein or theimmunogenic fragment of M2 protein; wherein (a) and (b) are linkedtogether, optionally via a linker.
 11. The method of claim 8, whereinthe fusion protein has an amino acid sequence as set forth in SEQ ID NO:36, 38, 42 or
 44. 12. The method of claim 1, wherein the fusion proteincomprises a linker.